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Hridoy M, Khan I, Ramanjulu M, Anthony P, Childers W, Nagar S, Korzekwa K. Mechanistic studies on pH-permeability relationships: Impact of the membrane polar headgroup region on pKa. Int J Pharm 2025; 673:125383. [PMID: 39993512 PMCID: PMC11949092 DOI: 10.1016/j.ijpharm.2025.125383] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2024] [Revised: 02/18/2025] [Accepted: 02/19/2025] [Indexed: 02/26/2025]
Abstract
Passive permeability through biological membranes requires partitioning of drug molecules into the lipid bilayer and subsequent permeation. Most drugs are weak acids or bases, making their ionization constants (pKa) critical for predicting permeation across biological barriers. The pH-partition hypothesis posits that only the uncharged form contributes to passive permeability, suggesting a proportional relationship between permeability and uncharged fraction. However, experimental pH-permeability profiles are not accurately predicted with neutral fractions calculated using aqueous pKa values. Interactions between charged solutes and phospholipids are expected to alter the pKa of drugs within the membrane. In this study, we use modeling and simulation and experimental partitioning in a biphasic surrogate phospholipid membrane system, diacetyl phosphatidylcholine (DAcPC) and n-hexane, to study pH dependent permeability. Models were constructed in which pKa values were either shifted or distributed around the aqueous pKa and the resulting neutral fractions were compared to pH-dependent permeabilities. For acids, models with shifted or distributed pKa values can explain pH-dependent permeabilities in Caco-2 cells, but these models were not predictive for bases. For partitioning studies, five probe drugs, two acidic (ketoprofen, tolbutamide), two basic (metoprolol, verapamil), and one neutral (diazepam), were partitioned between n-hexane and buffer or buffer-hydrated DAcPC at different pH values. The apparent pKa values in the surrogate phospholipid system (C6/DAcPC) were shifted from their aqueous pKa values. However, the resulting pKa values did not predict observed pH-dependent Caco-2 permeabilities. Models that decrease the pH-pKa difference improve permeability predictions for both bases and acids and use of a pKa shift or distribution can further improve predictions for acids.
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Affiliation(s)
- Md Hridoy
- Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA 19140, USA; Current address: Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
| | - Irfan Khan
- Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, Philadelphia, PA 19140, USA
| | - Mercy Ramanjulu
- Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA 19140, USA; Current address: Recombination Therapeutics, Pennsylvania Biotechnology Center, Doylestown, PA 18902, USA
| | - Paul Anthony
- Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA 19140, USA
| | - Wayne Childers
- Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA 19140, USA
| | - Swati Nagar
- Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA 19140, USA
| | - Ken Korzekwa
- Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA 19140, USA.
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Klitgaard M, Jacobsen J, Kristensen MN, Berthelsen R, Müllertz A. Characterizing interregional differences in the rheological properties and composition of rat small intestinal mucus. Drug Deliv Transl Res 2024; 14:3309-3320. [PMID: 38526635 PMCID: PMC11445339 DOI: 10.1007/s13346-024-01574-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/12/2024] [Indexed: 03/27/2024]
Abstract
The mucus layer in the small intestine is generally regarded as a barrier to drug absorption. However, the mucus layer is a complex system, and presently, only a few studies have been conducted to elucidate its physicochemical properties. The current study hypothesizes that the mucus layer contains solubility-enhancing surfactants and thus might aid the oral absorption of poorly water-soluble drugs. Mucus was sampled from sections of the small intestine of fasted rats to analyze the rheological properties and determine the mucus pH and concentrations of proteins and endogenous surfactants, i.e., bile salts, polar lipids, and neutral lipids. The mucus layer in the two proximal sections of the small intestine exhibited different rheological properties such as higher zero-shear viscosity and lower loss tangent and higher protein concentrations compared to all subsequent sections of the small intestine. The pH of the mucus layer was stable at ~ 6.5 throughout most of the small intestine, but increased to 7.5 in the ileum. The bile salt concentrations increased from the duodenum (16.0 ± 2.2 mM) until the mid jejunum (55.1 ± 9.5 mM), whereas the concentrations of polar lipids and neutral lipids decreased from the duodenum (17.4 ± 2.2 mM and 37.8 ± 1.6 mM, respectively) until the ileum (4.8 ± 0.4 mM and 10.7 ± 1.1 mM, respectively). In conclusion, the mucus layer of the rat small intestine contains endogenous surfactants at levels that might benefit solubilization and absorption of orally administered poorly water-soluble drugs.
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Affiliation(s)
- Mette Klitgaard
- Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark
| | - Jette Jacobsen
- Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark
| | - Maja Nørgaard Kristensen
- Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark
| | - Ragna Berthelsen
- Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark
| | - Anette Müllertz
- Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
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Shang H, Sun Y, Wang Z, Zhou Y, Yang H, Ci X, Cui T, Xia Y, Gu Y, Liao M, Li Q, Si D, Liu C. Intestinal absorption mechanism of rotundic acid: Involvement of P-gp and OATP2B1. JOURNAL OF ETHNOPHARMACOLOGY 2022; 289:115006. [PMID: 35051604 DOI: 10.1016/j.jep.2022.115006] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/23/2021] [Revised: 01/13/2022] [Accepted: 01/15/2022] [Indexed: 06/14/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Ilicis Rotundae Cortex (IRC), the dried barks of Ilex rotunda Thunb. (Aquifoliaceae), has been used for the prevention or treatment of colds, tonsillitis, dysentery, and gastrointestinal diseases in folk medicine due to its antibacterial and anti-inflammatory effects. However, there is no report about the intestinal absorption of major compounds that support traditional usage. AIM OF STUDY Considering the potential of rotundic acid (RA) - major biologically active pentacyclic triterpenes found in the IRC, this study was purposed to uncover the oral absorption mechanism of RA using in situ single-pass intestinal perfusion (SPIP) model, in vitro cell models (Caco-2, MDCKII-WT, MDCKII-MDR1, MDCKII-BCRP, and HEK293-OATP2B1 cells) and in vivo pharmacokinetics studies in rats. MATERIALS AND METHODS The molecular properties (solubility, lipophilicity, and chemical stability) and the effects of principal parameters (time, compound concentrations, pH, paracellular pathway, and the different intestinal segments) were analyzed by liquid chromatography-tandem mass spectrometry. The susceptibility of RA to various inhibitors, such as P-gp inhibitor verapamil, BCRP inhibitor Ko143, OATP 2B1 inhibitor rifampicin, and absorption enhancer EGTA were assessed. RESULTS RA was a compound with low water solubility (12.89 μg/mL) and strong lipophilicity (LogP = 4.1). RA was considered stable in all media during the SPIP and transport studies. The SPIP and cell experiments showed RA was moderate absorbed in the intestines and exhibited time, concentration, pH, and segment-dependent permeability. In addition, results from the cell model, in situ SPIP model as well as the in vivo pharmacokinetics studies consistently showed that verapamil, rifampicin, and EGTA might have significant effect on the intestinal absorption of RA. CONCLUSION The mechanisms of intestinal absorption of RA might involve multiple transport pathways, including passive diffusion, the participation of efflux (i.e., P-gp) and influx (i.e., OATP2B1) transporters, and paracellular pathways.
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Affiliation(s)
- Haihua Shang
- State Key Laboratory of Drug Delivery and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China; Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Peking Union Medical College & Chinese Academy of Medical Sciences, Tianjin, 300192, China
| | - Yinghui Sun
- Research Center of Bio-Technology, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China
| | - Ze Wang
- College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China
| | - Ying Zhou
- State Key Laboratory of Drug Delivery and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China
| | - Huajiao Yang
- State Key Laboratory of Drug Delivery and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China
| | - Xiaoyan Ci
- Research Center of Bio-Technology, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China
| | - Tao Cui
- Research Center of Bio-Technology, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China
| | - Yuanyuan Xia
- Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China; Research Unit for Drug Metabolism, Chinese Academy of Medical Sciences, Beijing, 100730, China
| | - Yuan Gu
- Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China; Research Unit for Drug Metabolism, Chinese Academy of Medical Sciences, Beijing, 100730, China
| | - Maoliang Liao
- State Key Laboratory of Drug Delivery and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China; Tianjin Ringpu Bio-technology Co., Ltd., Tianjin, 300308, China.
| | - Quansheng Li
- State Key Laboratory of Drug Delivery and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China
| | - Duanyun Si
- State Key Laboratory of Drug Delivery and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China; Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China; Research Unit for Drug Metabolism, Chinese Academy of Medical Sciences, Beijing, 100730, China
| | - Changxiao Liu
- State Key Laboratory of Drug Delivery and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China; Tianjin Institute of Pharmaceutical Research, Tianjin, 300000, China; Research Unit for Drug Metabolism, Chinese Academy of Medical Sciences, Beijing, 100730, China.
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Lee BL, Kuczera K, Lee KH, Childs EW, Jas GS. Unassisted N-acetyl-phenylalanine-amide transport across membrane with varying lipid size and composition: kinetic measurements and atomistic molecular dynamics simulation. J Biomol Struct Dyn 2020; 40:1445-1460. [PMID: 33034537 DOI: 10.1080/07391102.2020.1827037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
Biological membranes are essential to preserve structural integrity and regulate functional properties through the permeability of nutrients, pharmaceutical drugs, and neurotransmitters of a living cell. The movement of acetylated and amidated phenylalanine (NAFA) across 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane bilayers is investigated to probe physical transport. The rate of transport is measured experimentally applying parallel artificial membrane permeation assay (PAMPA). At the physiological temperature, 310 K, the measured time constants in the neutral pH were ∼6 h in DOPC and ∼3 h in POPC, while in a more acidic condition, at a pH 4.8, the time constants were ∼8 h in both lipids. Computationally, we have expanded our transport study of three aromatic dipeptides across a bilayer composed of DOPC18. In this study, we have examined the effects of lipid composition and bilayer size on the passive transport of NAFA by simulating the dipeptide in three different bilayers, with 50 DOPC lipids, 50 POPC lipids, and 40 POPC molecules. Specifically, atomistic molecular dynamics simulations with umbrella sampling were used to calculate the potential of mean force for the passive permeation of NAFA across the bilayers. Diffusion constants were then calculated by numerically solving the Smoluchowski equation. Permeability coefficients and mean first passage times were then calculated. Structural properties - Ramachandran plots, sidechain torsions, peptide insertion angles, radial distribution functions, and proximal peptide water molecules - were also examined to determine the effect of system size and lipid type. In terms of systems size, we observed a small decrease in the highest barrier of the potential of mean force and fewer sampled sidechain dihedral angle conformations with 40 versus 50 POPC lipids due to weaker membrane deformations within a smaller lipid bilayer. In terms of lipid type, DOPC contains two monounsaturated acyl chains compared to only one such acyl chain in POPC; therefore, DOPC bilayers are less ordered and more easily deformed, as seen by a much broader potential of mean force profile. The NAFA in DOPC lipid also transitioned to an internally hydrogen-bonded backbone conformation at lower membrane depths than in POPC. Similarly, as for other aromatic dipeptides, NAFA tends to insert into the membrane sidechain-first, remains mostly desolvated in the membrane center, and exhibits slow reorientations within the bilayer in both DOPC and POPC. With a joint experimental and computational study we have gained a new insight into the rate of transport and the underlying microscopic mechanism in different lipid bilayer conditions of the simplest hydrophobic aromatic dipeptide.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Brent L Lee
- Department of Chemistry, The University of Kansas, Lawrence, KS, USA
| | - Krzysztof Kuczera
- Department of Chemistry, The University of Kansas, Lawrence, KS, USA.,Department of Molecular Biosciences, The University of Kansas, Lawrence, KS, USA
| | - Kyung-Hoon Lee
- Department of Biology, Chowan University, Murfreesboro, NC, USA
| | - Ed W Childs
- Department of Surgery, Morehouse School of Medicine, Atlanta, GA, USA
| | - Gouri S Jas
- Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS, USA
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Faralli A, Shekarforoush E, Mendes AC, Chronakis IS. Enhanced Transepithelial Permeation of Gallic Acid and (-)-Epigallocatechin Gallate across Human Intestinal Caco-2 Cells Using Electrospun Xanthan Nanofibers. Pharmaceutics 2019; 11:pharmaceutics11040155. [PMID: 30939805 PMCID: PMC6523729 DOI: 10.3390/pharmaceutics11040155] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2018] [Revised: 03/17/2019] [Accepted: 03/20/2019] [Indexed: 12/16/2022] Open
Abstract
Electrospun xanthan polysaccharide nanofibers (X) were developed as an encapsulation and delivery system of the poorly absorbed polyphenol compounds, gallic acid (GA) and (−)-epigallocatechin gallate (EGCG). Scanning electron microscopy was used to characterize the electrospun nanofibers, and controlled release studies were performed at pH 6.5 and 7.4 in saline buffer, suggesting that the release of polyphenols from xanthan nanofibers follows a non-Fickian mechanism. Furthermore, the X-GA and X-EGCG nanofibers were incubated with Caco-2 cells, and the cell viability, transepithelial transport, and permeability properties across cell monolayers were investigated. Increases of GA and EGCG permeabilities were observed when the polyphenols were loaded into xanthan nanofibers, compared to the free compounds. The observed in vitro permeability enhancement of GA and EGCG was induced by the presence of the polysaccharide nanofibers, which successfully inhibited efflux transporters, as well as by opening tight junctions.
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Affiliation(s)
- Adele Faralli
- Nano-BioScience Research Group, DTU-Food, Technical University of Denmark, Kemitorvet, B202, 2800 Kgs. Lyngby, Denmark.
| | - Elhamalsadat Shekarforoush
- Nano-BioScience Research Group, DTU-Food, Technical University of Denmark, Kemitorvet, B202, 2800 Kgs. Lyngby, Denmark.
| | - Ana C Mendes
- Nano-BioScience Research Group, DTU-Food, Technical University of Denmark, Kemitorvet, B202, 2800 Kgs. Lyngby, Denmark.
| | - Ioannis S Chronakis
- Nano-BioScience Research Group, DTU-Food, Technical University of Denmark, Kemitorvet, B202, 2800 Kgs. Lyngby, Denmark.
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Faralli A, Shekarforoush E, Ajalloueian F, Mendes AC, Chronakis IS. In vitro permeability enhancement of curcumin across Caco-2 cells monolayers using electrospun xanthan-chitosan nanofibers. Carbohydr Polym 2019; 206:38-47. [DOI: 10.1016/j.carbpol.2018.10.073] [Citation(s) in RCA: 51] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2018] [Revised: 09/19/2018] [Accepted: 10/24/2018] [Indexed: 11/15/2022]
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Roos C, Dahlgren D, Sjögren E, Tannergren C, Abrahamsson B, Lennernäs H. Regional Intestinal Permeability in Rats: A Comparison of Methods. Mol Pharm 2017; 14:4252-4261. [PMID: 28920690 DOI: 10.1021/acs.molpharmaceut.7b00279] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Currently, the screening of new drug candidates for intestinal permeation is typically based on in vitro models which give no information regarding regional differences along the gut. When evaluation of intestinal permeability by region is undertaken, two preclinical rat models are commonly used, the Ussing chamber method and single-pass intestinal perfusion (SPIP). To investigate the robustness of in vivo predictions of human intestinal permeability, a set of four model compounds was systematically investigated in both these models, using tissue specimens and segments from the jejunum, ileum, and colon of rats from the same genetic strain. The influence of luminal pH was also determined at two pH levels. Ketoprofen had high and enalaprilat had low effective (Peff) and apparent (Papp) permeability in all three regions and at both pH levels. Metoprolol had high Peff in all regions and at both pHs and high Papp at both pHs and in all regions except the jejunum, where Papp was low. Atenolol had low Peff in all regions and at both pHs, but had high Papp at pH 6.5 and low Papp at pH 7.4. There were good correlations between these rat in situ Peff (SPIP) and human in vivo Peff determined previously for the same compounds by both intestinal perfusion of the jejunum and regional intestinal dosing. The results of this study indicate that both investigated models are suitable for determining the regional permeability of the intestine; however, the SPIP model seems to be the more robust and accurate regional permeability model.
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Affiliation(s)
- Carl Roos
- Department of Pharmacy, Uppsala University , Box 580, 751 23 Uppsala, Sweden
| | - David Dahlgren
- Department of Pharmacy, Uppsala University , Box 580, 751 23 Uppsala, Sweden
| | - Erik Sjögren
- Department of Pharmacy, Uppsala University , Box 580, 751 23 Uppsala, Sweden
| | - Christer Tannergren
- Pharmaceutical Technology and Development, AstraZeneca R&D , 431 83 Gothenburg, Sweden
| | - Bertil Abrahamsson
- Pharmaceutical Technology and Development, AstraZeneca R&D , 431 83 Gothenburg, Sweden
| | - Hans Lennernäs
- Department of Pharmacy, Uppsala University , Box 580, 751 23 Uppsala, Sweden
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Yusof SR, Abbott NJ, Avdeef A. Impact of capillary flow hydrodynamics on carrier-mediated transport of opioid derivatives at the blood-brain barrier, based on pH-dependent Michaelis-Menten and Crone-Renkin analyses. Eur J Pharm Sci 2017; 106:274-286. [PMID: 28614733 DOI: 10.1016/j.ejps.2017.06.016] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2017] [Revised: 04/10/2017] [Accepted: 06/09/2017] [Indexed: 12/01/2022]
Abstract
Most studies of blood-brain barrier (BBB) permeability and transport are conducted at a single pH, but more detailed information can be revealed by using multiple pH values. A pH-dependent biophysical model was applied to the mechanistic analysis of published pH-dependent BBB luminal uptake data from three opioid derivatives in rat: pentazocine (Suzuki et al., 2002a, 2002b), naloxone (Suzuki et al., 2010a), and oxycodone (Okura et al., 2008). Two types of data were processed: in situ brain perfusion (ISBP) and brain uptake index (BUI). The published perfusion data were converted to apparent luminal permeability values, Papp, and analyzed by the pCEL-X program (Yusof et al., 2014), using the pH-dependent Crone-Renkin equation (pH-CRE) to determine the impact of cerebrovascular flow on the Michaelis-Menten transport parameters (Avdeef and Sun, 2011). For oxycodone, the ISBP data had been measured at pH7.4 and 8.4. The present analysis indicates a 7-fold lower value of the cerebrovascular flow velocity, Fpf, than that expected in the original study. From the pyrilamine-inhibited data, the flow-corrected passive intrinsic permeability value was determined to be P0=398×10-6cm·s-1. The uptake data indicate that the neutral form of oxycodone is affected by a transporter at pH8.4. The extent of the cation uptake was less certain from the available data. For pentazocine, the brain uptake by the BUI method had been measured at pH5.5, 6.5, and 7.4, in a concentration range 0.1-40mM. Under similar conditions, ISBP data were also available. The pH-CRE determined values of Fpf from both methods were nearly the same, and were smaller than the expected value in the original publication. The transport of the cationic pentazocine was not fully saturated at pH5.5 at 40mM. The transport of the neutral species at pH7.4 appeared to reach saturation at 40mM pentazocine concentration, but not at 12mM. In the case of naloxone, a pH-dependent Michaelis-Menten equation (pH-MME) analysis of the data indicated a smooth sigmoidal transition from a higher capacity uptake process affecting cationic naloxone (pH5.0-7.0) to a lower capacity uptake process affecting the neutral drug (pH8.0-8.5), with cross-over point near pH7.4. Evidently, measurements at multiple pH values can reveal important information about both cerebrovascular flow and BBB transport kinetics.
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Affiliation(s)
- Siti R Yusof
- HICoE Centre for Drug Research, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
| | - N Joan Abbott
- King's College London, Institute of Pharmaceutical Science, Franklin Wilkins Building, 150 Stamford St., London SE1 9NH, UK
| | - Alex Avdeef
- in-ADME Research, 1732 First Avenue, #102, New York, NY 10128, USA.
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Roos C, Dahlgren D, Berg S, Westergren J, Abrahamsson B, Tannergren C, Sjögren E, Lennernäs H. In Vivo Mechanisms of Intestinal Drug Absorption from Aprepitant Nanoformulations. Mol Pharm 2017; 14:4233-4242. [DOI: 10.1021/acs.molpharmaceut.7b00294] [Citation(s) in RCA: 42] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Carl Roos
- Department
of Pharmacy, Uppsala University, 752 36 Uppsala, Sweden
| | - David Dahlgren
- Department
of Pharmacy, Uppsala University, 752 36 Uppsala, Sweden
| | | | - Jan Westergren
- Wendelsbergs beräkningskemi AB, Kyrkvägen 7B, 435 35 Mölnlycke, Sweden
| | | | | | - Erik Sjögren
- Department
of Pharmacy, Uppsala University, 752 36 Uppsala, Sweden
| | - Hans Lennernäs
- Department
of Pharmacy, Uppsala University, 752 36 Uppsala, Sweden
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10
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Intestinal absorption characteristics of imperialine: in vitro and in situ assessments. Acta Pharmacol Sin 2015; 36:863-73. [PMID: 26051111 DOI: 10.1038/aps.2015.27] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/02/2014] [Accepted: 02/05/2015] [Indexed: 02/05/2023]
Abstract
AIM Imperialine is an effective compound in the traditional Chinese medicine chuanbeimu (Bulbus Fritillariae Cirrhosae) that has been used as antitussive/expectorant in a clinical setting. In this study we investigated the absorption characteristics of imperialine in intestinal segments based on an evaluation of its physicochemical properties. METHODS Caco-2 cells were used to examine uptake and transport of imperialine in vitro, and a rat in situ intestinal perfusion model was used to characterize the absorption of imperialine. The amount of imperialine in the samples was quantified using LC-MS/MS. RESULTS The aqueous solubility and oil/water partition coefficient of imperialine were determined. This compound demonstrated a relatively weak alkalinity with a pKa of 8.467±0.028. In Caco-2 cells, the uptake of imperialine was increased with increasing pH in medium, but not affected by temperature. The apparent absorptive and secretive coefficient was (8.39±0.12)×10(-6) cm/s and (7.78±0.09)×10(-6) cm/s, respectively. Furthermore, neither the P-glycoprotein inhibitor verapamil nor Niemann-Pick C1-Like 1 transporter inhibitor ezetimibe affected the absorption and secretion of imperialine in vitro. The in situ intestinal perfusion study showed that the absorption parameters of imperialine varied in 4 intestinal segments (duodenum, jejunum, ileum and colon) with the highest ones in the colon, where a greater number of non-ionized form of imperialine was present. CONCLUSION The intestinal absorptive characteristics of imperialine are closely related to its physicochemical properties. The passive membrane diffusion dominates the intestinal absorption of imperialine.
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11
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Posada MM, Smith DE. Relevance of PepT1 in the intestinal permeability and oral absorption of cefadroxil. Pharm Res 2013; 30:1017-25. [PMID: 23224978 PMCID: PMC3596500 DOI: 10.1007/s11095-012-0937-8] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2012] [Accepted: 11/11/2012] [Indexed: 01/04/2023]
Abstract
PURPOSE To determine the contribution of intestinal PepT1 on the permeability and oral absorption of the β-lactam antibiotic drug cefadroxil. METHODS The effective permeability (P eff ) of cefadroxil was evaluated in wild-type and PepT1 knockout mice following in situ single-pass intestinal perfusions. The plasma concentration-time profiles of cefadroxil were also examined after oral gavage. RESULTS The P eff (cm/s) of cefadroxil in wild-type mice was 0.49 × 10(-4) in duodenum, 0.80 × 10(-4) in jejunum, 0.88 × 10(-4) in ileum and 0.064 × 10(-4) in colon. The P eff (cm/s) in PepT1 knockout mice was significantly reduced in small intestine, but not in colon, as shown by values of 0.003 × 10(-4), 0.090 × 10(-4), 0.042 × 10(-4) and 0.032 × 10(-4), respectively. Jejunal uptake of cefadroxil was saturable (Km = 2-4 mM) and significantly attenuated by the sodium-proton exchange inhibitor 5-(N,N-dimethyl)amiloride. Jejunal permeability of cefadroxil was not affected by L-histidine, glycine, cephalothin, p-aminohippurate or N-methylnicotinamide. In contrast, cefadroxil permeability was significantly reduced by glycylproline, glycylsarcosine, or cephalexin. Finally, PepT1 ablation resulted in 23-fold reductions in peak plasma concentrations and 14-fold reductions in systemic exposure of cefadroxil after oral dosing. CONCLUSIONS The findings are definitive in demonstrating that PepT1 is the major transporter responsible for the small intestinal permeability of cefadroxil as well as its enhanced oral drug performance.
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Affiliation(s)
- Maria M Posada
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-5633, USA
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12
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Rong Z, Xu Y, Zhang C, Xiang D, Li X, Liu D. Evaluation of intestinal absorption of amtolmetin guacyl in rats: Breast cancer resistant protein as a primary barrier of oral bioavailability. Life Sci 2013; 92:245-51. [DOI: 10.1016/j.lfs.2012.12.010] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2012] [Revised: 10/05/2012] [Accepted: 12/17/2012] [Indexed: 02/06/2023]
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Kang MJ, Kim HS, Jeon HS, Park JH, Lee BS, Ahn BK, Moon KY, Choi YW. In situ intestinal permeability and in vivo absorption characteristics of olmesartan medoxomil in self-microemulsifying drug delivery system. Drug Dev Ind Pharm 2012; 38:587-596. [PMID: 21988221 DOI: 10.3109/03639045.2011.619194] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
To characterize the intestinal absorption behavior of olmesartan medoxomil (OLM) and to evaluate the absorption-improving potential of a self-microemulsifying drug delivery system (SMEDDS), we performed in situ single-pass intestinal perfusion (SPIP) and in vivo pharmacokinetic studies in rats. The SPIP study revealed that OLM is absorbed throughout whole intestinal regions, favoring proximal segments, at drug levels of 10-90 μM. The greatest value for effective permeability coefficient (P(eff)) was 11.4 × 10(-6) cm/s in the duodenum (90 μM); the lowest value was 2.9 × 10(-6) cm/s in the ileum (10 μM). A SMEDDS formulation consisting of Capryol 90, Labrasol, and Transcutol, which has a droplet size of 200 nm and self-dispersion time of 21 s, doubled upper intestinal permeability of OLM. The SMEDDS also improved oral bioavailability of OLM in vivo: a 2.7-fold increase in the area under the curve (AUC) with elevated maximum plasma concentration (C(max)) and shortened peak time (T(max)) compared to an OLM suspension. A strong correlation (r(2) = 0.955) was also found between the in situ jejunal P(eff) and the in vivo AUC values. Our study illustrates that the SMEDDS formulation holds great potential as an alternative to increased oral absorption of OLM.
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Affiliation(s)
- Myung J Kang
- Division of Pharmaceutical Sciences, College of Pharmacy, Chung-Ang University, Dongjak-gu, Seoul, Korea
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14
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15
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Jappar D, Wu SP, Hu Y, Smith DE. Significance and regional dependency of peptide transporter (PEPT) 1 in the intestinal permeability of glycylsarcosine: in situ single-pass perfusion studies in wild-type and Pept1 knockout mice. Drug Metab Dispos 2010; 38:1740-6. [PMID: 20660104 PMCID: PMC2957162 DOI: 10.1124/dmd.110.034025] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2010] [Accepted: 07/21/2010] [Indexed: 01/07/2023] Open
Abstract
The purpose of this study was to evaluate the role, relevance, and regional dependence of peptide transporter (PEPT) 1 expression and function in mouse intestines using the model dipeptide glycylsarcosine (GlySar). After isolating specific intestinal segments, in situ single-pass perfusions were performed in wild-type and Pept1 knockout mice. The permeability of [(3)H]GlySar was measured as a function of perfusate pH, dipeptide concentration, potential inhibitors, and intestinal segment, along with PEPT1 mRNA and protein. We found the permeability of GlySar to be saturable (K(m) = 5.7 mM), pH-dependent (maximal value at pH 5.5), and specific for PEPT1; other peptide transporters, such as PHT1 and PHT2, were not involved, as judged by the lack of GlySar inhibition by excess concentrations of histidine. GlySar permeabilities were comparable in the duodenum and jejunum of wild-type mice but were much larger than that in ileum (approximately 2-fold). A PEPT1-mediated permeability was not observed for GlySar in the colon of wild-type mice (<10% residual uptake compared to proximal small intestine). Moreover, GlySar permeabilities were very low and not different in the duodenum, jejunum, ileum, and colon of Pept1 knockout mice. Functional activity of intestinal PEPT1 was confirmed by real-time polymerase chain reaction and immunoblot analyses. Our findings suggest that a loss of PEPT1 activity (e.g., due to polymorphisms, disease, or drug interactions) should have a major effect in reducing the intestinal absorption of di-/tripeptides, peptidomimetics, and peptide-like drugs.
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Affiliation(s)
- Dilara Jappar
- University of Michigan, 4742 Medical Sciences II, 1150 W Medical Center Drive, Ann Arbor, MI 48109-5633, USA
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16
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Ma K, Hu Y, Smith DE. Peptide transporter 1 is responsible for intestinal uptake of the dipeptide glycylsarcosine: studies in everted jejunal rings from wild-type and Pept1 null mice. J Pharm Sci 2010; 100:767-74. [PMID: 20862774 DOI: 10.1002/jps.22277] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2010] [Revised: 05/21/2010] [Accepted: 05/24/2010] [Indexed: 01/02/2023]
Abstract
The purpose of this study was to determine the relative importance of peptide transporter 1 (PEPT1) in the uptake of peptides/mimetics from mouse small intestine, using glycylsarcosine (GlySar). After isolating jejunal tissue from wild-type and Pept1 null mice, 2 cm intestinal segments were everted and mounted on glass rods for tissue uptake studies. [(14)C]GlySar (4 μM) was studied as a function of time, temperature, sodium and pH, concentration, and potential inhibitors. Compared with wild-type animals, Pept1 null mice exhibited a 78% reduction in GlySar uptake at pH 6.0 at 37°C. GlySar uptake showed pH dependence, with peak values between pH 6.0 and 6.5 in wild-type animals, whereas no such tendency was observed in Pept1 null mice. GlySar exhibited Michaelis-Menten uptake kinetics and a minor nonsaturable component in wild-type animals. In contrast, GlySar uptake occurred only by a nonsaturable process in Pept1 null mice. GlySar uptake was significantly inhibited by dipeptides, aminocephalosporins, angiotensin-converting enzyme inhibitors, and the antiviral prodrug valacyclovir; these inhibitors had little, if any, effect on the uptake of GlySar in Pept1 null mice. The findings demonstrate that PEPT1 plays a critical role in the uptake of GlySar in jejunum and suggest that PEPT1 is the major transporter responsible for the intestinal absorption of small peptides.
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Affiliation(s)
- Katherine Ma
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, USA
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17
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Sugano K, Kansy M, Artursson P, Avdeef A, Bendels S, Di L, Ecker GF, Faller B, Fischer H, Gerebtzoff G, Lennernaes H, Senner F. Coexistence of passive and carrier-mediated processes in drug transport. Nat Rev Drug Discov 2010; 9:597-614. [PMID: 20671764 DOI: 10.1038/nrd3187] [Citation(s) in RCA: 460] [Impact Index Per Article: 30.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The permeability of biological membranes is one of the most important determinants of the pharmacokinetic processes of a drug. Although it is often accepted that many drug substances are transported across biological membranes by passive transcellular diffusion, a recent hypothesis speculated that carrier-mediated mechanisms might account for the majority of membrane drug transport processes in biological systems. Based on evidence of the physicochemical characteristics and of in vitro and in vivo findings for marketed drugs, as well as results from real-life discovery and development projects, we present the view that both passive transcellular processes and carrier-mediated processes coexist and contribute to drug transport activities across biological membranes.
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Affiliation(s)
- Kiyohiko Sugano
- Pfizer, Research Formulation, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK.
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18
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Sugano K. Aqueous Boundary Layers Related to Oral Absorption of a Drug: From Dissolution of a Drug to Carrier Mediated Transport and Intestinal Wall Metabolism. Mol Pharm 2010; 7:1362-73. [DOI: 10.1021/mp1001119] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Kiyohiko Sugano
- Global Research & Development, Sandwich Laboratories, Research Formulation, Pfizer Inc., CT13 9NJ, Sandwich, Kent, U.K
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19
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Avdeef A, Tam KY. How well can the Caco-2/Madin-Darby canine kidney models predict effective human jejunal permeability? J Med Chem 2010; 53:3566-84. [PMID: 20373811 DOI: 10.1021/jm901846t] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
The study aimed to predict effective human jejunal permeability (P(eff)) using a biophysical model based on parametrized paracellular, aqueous boundary layer, and transcellular permeabilities, and the villus-fold surface area expansion factor (k(VF)). Published human jejunal data (119 P(eff), 53 compounds) were analyzed by a regression procedure incorporating a dual-pore size paracellular model. Transcellular permeability, scaled by k(VF), was equated to that of Caco-2 at pH 6.5. The biophysical model predicted human jejunal permeability data within the experimental uncertainty. This investigation revealed several surprising predictions: (i) many molecules permeate predominantly (but not exclusively) by the paracellular route, (ii) the aqueous boundary layer thickness in the intestinal perfusion experiments is larger than expected, (iii) the mucosal surface area in awake humans is apparently nearly entirely accessible to drug absorption, and (iv) the relative "leakiness" of the human jejunum is not so different from that observed in a number of published Caco-2 studies.
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Affiliation(s)
- Alex Avdeef
- pION Inc., 5 Constitution Way, Woburn, Massachusetts 01801, USA.
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Lafforgue G, Arellano C, Vachoux C, Woodley J, Philibert C, Dupouy V, Bousquet-Mélou A, Gandia P, Houin G. Oral absorption of ampicillin: role of paracellular route vs. PepT1 transporter. Fundam Clin Pharmacol 2008; 22:189-201. [PMID: 18353114 DOI: 10.1111/j.1472-8206.2008.00572.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The beta-lactam antibiotic ampicillin has a relatively poor oral bioavailability in animals and man (30-40%), and its widespread agricultural use in livestock may be contributing to the emergence of antibiotic resistance in the environment. The aim of this study was to define the absorption mechanism by which ampicillin crosses the small intestinal epithelium. The improved rat everted gut sac system was used, with an emphasis on the role of the PepT1 transporter. The absorption kinetics, effects of pH and the use of competitive substrates failed to provide any substantive evidence that the transporter played a major role in ampicillin absorption. Ethylenediaminetetraacetic acid enhanced the absorption, and tissue levels remained low, suggesting that paracellular transport was predominant. pH and competition studies with glycylsarcosine, the widely used PepT1 substrate, also failed to show any transporter activity. Despite evidence from studies with Caco-2 cells that beta-lactam antibiotics are transported by the PepT1 transporter in rat small intestine, the results rather suggest that paracellular diffusion is the major mechanism of absorption, at least for beta-lactam antibiotics with poor bioavailability, such as ampicillin. We suggest that the use of Caco-2 cells underestimates the role of the paracellular route in the absorption of hydrophilic drugs in vivo, and may exaggerate the role of influx transporters.
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Affiliation(s)
- Guylène Lafforgue
- Laboratoire Cinétique des Xénobiotiques, Faculté des Sciences Pharmaceutiques, 35 Chemin des Maraîchers, 31062 Toulouse cedex 4, France
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21
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Xie YS, Ren XL, Pan GX, Gao XM, Liu CX. The assessment of absorption of periplocin in situ via intestinal perfusion of rats by HPLC. Biomed Chromatogr 2008; 22:196-201. [PMID: 18059065 DOI: 10.1002/bmc.914] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Periplocin is an important compound of Cortex Periplocae, which shows poor absorption when administered orally. The effective intestinal permeability of periplocin was investigated using single-pass intestinal perfusion technique in male Wistar rats. SPIP was performed in rat jejunum. The samples of perfusate were collected at the designated time points after rat intestinal perfusion and analyzed by HPLC. The specificity of this method was demonstrated by the absence of interference of the drug peak with the intestinal sac artifacts and the components of the KRB solution. Recovery studies, as well as the intra-day and inter-day variations, were within statistical limits. This technique was applied to the study of the intestinal absorption of periplocin. The determined fraction absorbed (F(a)) of periplocin was 0.151 +/- 0.072 (n = 6) at a concentration of 6 microg/mL; the absorption rate constant (K(a)) was 0.0102 +/- 0.0039/min and the effective permeability coefficient (P(eff)) was 0.0021 +/- 0.0012 cm/min. These data suggest that periplocin has high permeability and might be absorbed in rat intestine.
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Affiliation(s)
- Yue-Sheng Xie
- Pharmacokinetics Laboratory of Shenyang Pharmaceutical University, 103 Wenhua Rd, Shenyang 110016, People's Republic of China
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22
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Avdeef A, Kansy M, Bendels S, Tsinman K. Absorption-excipient-pH classification gradient maps: sparingly soluble drugs and the pH partition hypothesis. Eur J Pharm Sci 2007; 33:29-41. [PMID: 17983735 DOI: 10.1016/j.ejps.2007.09.009] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2007] [Revised: 07/27/2007] [Accepted: 09/17/2007] [Indexed: 11/27/2022]
Abstract
This study of sparingly soluble model drugs assesses (a) how pH and the aqueous boundary layer factors may affect in vitro and in vivo absorption, (b) to what extent single excipients (sodium taurocholate, hydroxypropyl-beta-cyclodextrin, KCl, propylene glycol, methylpyrrolidone, and polyethylene glycol 400) can mitigate adverse absorption effects, and (c) how a novel rank-order visualization tool can be applied in high-throughput screening to identify promising single-excipient effects on the absorption potential of test compounds. The products of accurately measured solubility and artificial-membrane permeability (PAMPA) values at pH 5.0, 6.2, and 7.4, fully taking into account factors such as aqueous boundary layer resistance, membrane retention, and the formation of drug dimers and trimers, were used to define a flux function. A "self-organized" data visualization tool based on the flux function was mined for the promising excipient-drug combinations. In excipient-free solutions, most of the compounds studied formed aggregates. The presence of an excipient predominantly lowered permeability, but most often not by the same amount as solubility was elevated. The compounds with absorption potential most helped by excipients were: clotrimazole>griseofulvin>progesterone>dipyridamole>glibenclamide>mefenamic acid>butacaine>astemizole. The HP-beta-CD effect observed for albendazole and glibenclamide appeared to follow Cmax trends in published pharmacokinetics studies. A surprising outcome of the in vitro measurements was that the classical pH Partition Hypothesis can be "inverted" in its monotonicity by sparingly soluble compounds.
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Affiliation(s)
- Alex Avdeef
- pION INC, 5 Constitution Way, Woburn, MA 01801 USA.
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23
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Nguyen TV, Fleisher D, Smith DE. In Vivo Effects of Glycyl-Glutamate and Glycyl-Sarcosine on Gabapentin Oral Absorption in Rat. Pharm Res 2007; 24:1538-43. [PMID: 17380260 DOI: 10.1007/s11095-007-9272-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2006] [Accepted: 02/12/2007] [Indexed: 10/23/2022]
Abstract
PURPOSE The objective of this study was to evaluate the in vivo consequences of glycyl-glutamate coadministration on gabapentin oral absorption. METHODS Rats were administered gabapentin (10 mg/kg plus radiotracer) by gastric gavage, in the absence and presence of dipeptides, and by intravenous administration. Serial blood samples were obtained over 6 h and the pharmacokinetics of gabapentin were determined by noncompartmental analysis. RESULTS Glycyl-glutamate coadministration increased the Cmax of gabapentin by 86% as compared to gabapentin alone. In agreement, the oral absorption of gabapentin, relative to the intravenous dose, was 79% after glycyl-glutamate loading but only 47% when drug was administered alone. However, when glycyl-sarcosine was added to the orally administered admixture of gabapentin plus glycyl-glutamate, values for Cmax and AUC(0-6 h) reverted back to that of control. In contrast, the tmax and terminal half-life of gabapentin did not change after oral dosing for all treatments. CONCLUSIONS These findings are unique in demonstrating that under physiologic, in vivo conditions, the luminal presence of glycyl-glutamate could dramatically enhance the Cmax and AUC(0-6 h) of gabapentin. The results are consistent with previous in situ intestinal perfusion studies in rat, and establish a functional interaction between the activities of PEPT1 and amino acid exchangers.
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Affiliation(s)
- Theresa V Nguyen
- Department of Pharmaceutical Sciences, The University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
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24
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Nguyen TV, Smith DE, Fleisher D. PEPT1 Enhances the Uptake of Gabapentin via Trans-Stimulation of b0,+ Exchange. Pharm Res 2006; 24:353-60. [PMID: 17192834 DOI: 10.1007/s11095-006-9155-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2006] [Accepted: 08/28/2006] [Indexed: 10/23/2022]
Abstract
PURPOSE The aims of this study were (1) to determine whether amino acid and dipeptide loading can improve the effective permeability of gabapentin and (2) to characterize the underlying mechanism that is responsible for this interaction. MATERIALS AND METHODS An in situ single-pass rat intestinal perfusion model was used to assess the effective permeability of gabapentin in rat, in the absence and presence of cellular loading by amino acid and dipeptide mixtures. RESULTS Compared to gabapentin alone, cellular loading with amino acid and dipeptide mixtures significantly improved the effective permeability of gabapentin by 46-79% in jejunum and by 67-72% in ileum (p < or = 0.01). However, coperfusion of glycylsarcosine (i.e., PEPT1 substrate), methionine sulfoximine (i.e., glutamine synthase inhibitor), or lysine and arginine (i.e., b(0,+) substrates) with the amino acid and dipeptide mixtures compromised the intestinal uptake of gabapentin. CONCLUSIONS These findings demonstrate, for the first time, a direct relationship between the PEPT1-mediated uptake of a dipeptide and the trans-stimulated uptake of gabapentin (an amino acid-like drug) through the transport system b(0,+).
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Affiliation(s)
- Theresa V Nguyen
- Deparment of Pharmaceutical Sciences, College of Pharmacy, The University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109-1065, USA.
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25
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Abstract
AIM: To characterize the intestinal transport and mechanism of metformin in rats and to investigate whether or not metformin is a substrate for P-glycoprotein (P-gp).
METHODS: The effective intestinal permeability of metformin was investigated using single-pass intestinal perfusion (SPIP) technique in male Waster rats. SPIP was performed in three isolated intestinal segments (duodenum, jejunum and ileum) at the same concentration of metformin (50 μg/mL) to test if the intestinal transport of metformin exhibited site-dependent changes, and in a same isolated intestinal segment (duodenal segment) at three different concentrations of metformin (10, 50, 200 μg/mL) to test if the intestinal transport of metformin exhibited concentration-dependent changes. Besides, P-gp inhibitor verapamil (400 μg/mL) was co-perfused with metformin (50 μg/mL) in the duodenum segment to find out if the intestinal absorption of metformin was affected by P-gp exiting along the gastrointestinal track. Stability studies were conducted to ensure that the loss of metformin could be attributed to intestinal absorption.
RESULTS: The effective permeability values (Peff) of metformin in the jejunum and ileum at 50 μg/mL were significantly lower than those in the duodenum at the same concentration. Besides, Peff values in the duodenum at high concentration (200 μg/mL) were found to be significantly lower than those at low and medium concentrations (10 and 50 μg/mL). Moreover the co-perfusion with verapamil did not increase the Peff value of metformin at 50 μg/mL in the duodenum.
CONCLUSION: Metformin could be absorbed from the whole intestine, with the main absorption site at duodenum. This concentration-dependent permeability behavior in the duodenum indicates that metformin is transported by both passive and active carrier-mediated saturable mechanism. The Peff value can not be increased by co-perfusion with verapamil, indicating that absorption of metformin is not efficiently transported by P-gp in the gut wall. Furthermore metformin is neither a substrate nor an inducer of P-gp. Based on the Peff values obtained in the present study and using established relationships, the human fraction dose absorbed for metformin is estimated to be 74%-90% along human intestine.
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Affiliation(s)
- Nai-Ning Song
- School of Chemical Engineering and Technology, Tianjin University, China
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26
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Zakeri-Milani P, Barzegar-Jalali M, Tajerzadeh H, Azarmi Y, Valizadeh H. Simultaneous determination of naproxen, ketoprofen and phenol red in samples from rat intestinal permeability studies: HPLC method development and validation. J Pharm Biomed Anal 2005; 39:624-30. [PMID: 15899568 DOI: 10.1016/j.jpba.2005.04.008] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2004] [Revised: 04/10/2005] [Accepted: 04/11/2005] [Indexed: 10/25/2022]
Abstract
A simple reversed-phase high performance liquid chromatographic method with UV detection at 270 nm was developed for simultaneous quantitation of ketoprofen and naproxen sodium along with phenol red as a non-absorbable marker for in situ permeability studies. The mobile phase was a mixture of 20% methanol, 28% of acetonitrile, 52% water and 0.4 ml triethylamine (adjusted to pH 3.2 using orthophosphoric acid). Analysis was run at a flow of 1.5 ml/min with a 20 min run time. The calibration curves were linear for all three compounds (r>0.999) across the concentration range of 15.6-250 microg/ml with a limit of quantitation of 0.3, 0.25 and 0.2 ng/ml for naproxen, ketoprofen and phenol red, respectively. The coefficient of variation for intra-assay and inter-assay precision was less than or equal to 5.3% and the accuracy was between 95.36 and 101.6%. Using the SPIP technique and the suggested HPLC method for sample analysis, the mean values of 1.17e(-4) (+/-0.28) cm/s and 0.97e(-4) (+/-0.2) cm/s were obtained for naproxen and ketoprofen, respectively.
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Affiliation(s)
- Parvin Zakeri-Milani
- Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
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27
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Lee KJ, Johnson N, Castelo J, Sinko PJ, Grass G, Holme K, Lee YH. Effect of experimental pH on the in vitro permeability in intact rabbit intestines and Caco-2 monolayer. Eur J Pharm Sci 2005; 25:193-200. [PMID: 15911214 DOI: 10.1016/j.ejps.2005.02.012] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2004] [Revised: 02/15/2005] [Accepted: 02/16/2005] [Indexed: 11/30/2022]
Abstract
The effect of experimental (apical) pH on absorptive permeability (Pe) was investigated in animal intestinal tissues and Caco-2 cell monolayers to examine whether the introduction of physiological pH such as 6.5 relates to the better prediction of animal intestinal Pe. Transport studies were conducted in a 24-well transwell for Caco-2 and diffusion chambers for rabbit intestinal permeability. Twenty-four test compounds were chosen (seven acidic, seven basic, eight neutral, and two zwitterionic) and Pe was measured at a 100microM donor concentration with two apical pHs, Krebs Bicarbonate Ringer's buffer (pH 7.4) and 4-morpholineethanesulfonic acid (MES) buffer (pH 6.5). Samples were collected over a 90-min interval and analyzed by LC/UV, LC/MS, or LSC. Upon the apical pH change from 7.4 to 6.5, Caco-2 Pe of acidic and basic compounds changed significantly, whereas rabbit intestinal Pe did not change possibly by the presence of mucous layer. When the intestinal Pe was correlated with pH 6.5 or 7.4 Caco-2 Pe, the correlation of pH 6.5 duodenum and jejunum Pe with pH 6.5 Caco-2 Pe was very poor. However, pH 7.4 Caco-2 Pe correlated relatively well with pH 6.5 duodenum and jejunum Pe and pH 7.4 ileum and colon Pe. The results suggested that pH 7.4 Caco-2 Pe is a good qualitative predictor for physiological intestinal permeability from duodenum to colon.
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28
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Zakelj S, Legen I, Veber M, Kristl A. The influence of buffer composition on tissue integrity during permeability experiments "in vitro". Int J Pharm 2004; 272:173-80. [PMID: 15019080 DOI: 10.1016/j.ijpharm.2003.12.014] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2003] [Revised: 12/02/2003] [Accepted: 12/10/2003] [Indexed: 11/28/2022]
Abstract
A well-balanced incubation saline is necessary for permeability experiments with the rat jejunal tissue in the diffusion chambers. At the same time the investigated substance must be chemically stable and sufficiently soluble in this incubation saline. To investigate whether the absence of some ions in incubation salines influences the tissue viability and integrity or the diffusional characteristics of the epithelial membrane the electrical parameters were monitored and the permeability of fluorescein and acyclovir was evaluated during the experiments in side-by-side diffusion chambers. Our results show that the tissue integrity and viability are seriously impaired when Ca(2+) and Mg(2+)-free conditions are applied on both sides of the diffusion chambers, but not when only mucosal or only serosal side is Ca(2+) and Mg(2+)-free. Bicarbonate-free incubation salines can also alter the measured apparent permeability coefficients even though the tissue viability and integrity do not change. This change in the apparent permeability is most likely due to a change in the pH of the mucosal surface and can be prevented if the buffer capacity of the incubation saline is increased.
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Affiliation(s)
- Simon Zakelj
- Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia
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29
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Legen I, Kristl A. pH and energy dependent transport of ketoprofen across rat jejunum in vitro. Eur J Pharm Biopharm 2003; 56:87-94. [PMID: 12837486 DOI: 10.1016/s0939-6411(03)00039-0] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The aim of this study was to elucidate transport mechanisms of ketoprofen (monocarboxylic acid with pK(a) 4.6) across rat jejunum in vitro using side-by-side diffusion cells. When the tissue was incubated on the mucosal and serosal sides with buffer of pH 7.51 (pH of the mucosal surface was 7.08), ketoprofen permeated faster in the mucosal-to-serosal than in the opposite direction. No asymmetry in transport was observed when 2 mM mucus disrupting agent 1,4-dithio-DL-threitol (pH of the mucosal surface increased to 7.21) was added to the mucosal side. Mucosal-to-serosal permeability of ketoprofen increased three times when the pH of the incubation medium was changed from 8.06 (pH of the mucosal surface was 7.34) to 6.07 (pH of the mucosal surface was 5.95), while no pH dependence was found under ATP-depletion caused by sodium azide. In the ketoprofen concentration range from 0.125 to 5 mM no saturation of transport was observed. Moreover, ketoprofen transport was not changed in the presence of 2 mM benzoate, 10 and 20 mM acetate, 20 mM L-lactate (substrates for monocarboxylate transporter 1, MCT1) and 1 mM alpha-cyano-4-hydroxy-cinnamic acid (an inhibitor of MCT1). These results indicate that ketoprofen is transported across rat jejunum in vitro by pH and energy dependent transport mechanisms, and most probably not by MCT1.
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Affiliation(s)
- Igor Legen
- Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
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Legen I, Kristl A. Factors affecting the microclimate pH of the rat jejunum in ringer bicarbonate buffer. Biol Pharm Bull 2003; 26:886-9. [PMID: 12808306 DOI: 10.1248/bpb.26.886] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
General characteristics of the microclimate layer on the mucosal surface of the rat jejunum incubated in bicarbonate buffers were investigated in vitro using pH sensitive flat membrane microelectrode. Jejunal surface microclimate (JSM) pH changed from 7.30+/-0.03 to 5.83+/-0.04 when the incubation buffer pH decreased from 8.03+/-0.02 to 6.12+/-0.01. Treatment of the mucosal side with mucolytic substances L-cysteine (1% (wt/v)) and 1,4-dithio-DL-threitol (2 mM) significantly (p<0.01) increased JSM pH. Respiratory chain inhibitor, sodium azide (10 mM) also significantly (p<0.05) increased JSM pH. D-Glucose (10 mM) at the mucosal side markedly (p<0.05) decreased JSM pH, which was attenuated by Na(+)/H(+) exchange inhibitor, amiloride (1 mM). Amiloride had no effect on JSM pH when D-glucose was not present at the mucosal side. In contrast to previous observations using bicarbonate free incubation buffers, we have demonstrated that JSM pH is not a constant value, but is dependent on pH of the incubation buffer. Additionally, Na(+)/H(+) exchanger does not contribute to acidic properties of JSM, when there is no D-glucose in the bicarbonate incubation buffer at the mucosal side of the tissue. In conclusion, we suggest that the bicarbonate buffers which are more close to in vivo situation than bicarbonate free buffers should be preferable incubation media when examining JSM.
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Affiliation(s)
- Igor Legen
- Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia
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Legen I, Zakelj S, Kristl A. Polarised transport of monocarboxylic acid type drugs across rat jejunum in vitro: the effect of mucolysis and ATP-depletion. Int J Pharm 2003; 256:161-6. [PMID: 12695022 DOI: 10.1016/s0378-5173(03)00073-5] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The transport characteristics of monocarboxylic acid type drugs (ketoprofen, ibuprofen and gemfibrozil) across the excised jejunal segments and artificial (octanol impregnated) membrane in side-by-side diffusion cells were studied. All three model drugs permeated faster across the intestinal tissue in the mucosal-to-serosal direction than in the opposite direction. No polarised transport of tested drugs was observed when the mucosal side of the intestine was treated with mucus disrupting agent, L-cysteine 1% (w/v), which significantly increased the microclimate pH at the mucosal surface of the intestine. Similar effects on the transport characteristics of model drugs and microclimate pH were observed when metabolic inhibitor, sodium azide (10mM), was present in the incubation medium. Furthermore, the direction of proton gradient across the artificial membrane was shown to significantly influence the transport of model drugs across this membrane. The results of this study indicate that the inwardly directed proton gradient maintained by the acidic microclimate pH at the intestinal surface could be considered as a driving force for the transport of monocarboxylic acid type drugs across the intestinal epithelia and could explain rapid absorption of these drugs after oral application.
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Affiliation(s)
- I Legen
- Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000, Ljubljana, Slovenia
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32
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Salphati L, Childers K, Pan L, Tsutsui K, Takahashi L. Evaluation of a single-pass intestinal-perfusion method in rat for the prediction of absorption in man. J Pharm Pharmacol 2001; 53:1007-13. [PMID: 11480535 DOI: 10.1211/0022357011776252] [Citation(s) in RCA: 88] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022]
Abstract
Prediction of the fraction of dose absorbed from the intestine (Fa) in man is essential in the early drug discovery stage. In-vitro assays in Caco-2 and MDCK cells are routinely used for that purpose, and their predictive value has been reported. However, in-situ techniques might provide a more accurate estimation of Fa. In this study, we evaluated a single-pass intestinal-perfusion (SPIP) method in the rat for its use in the prediction of absorption in man and compared it with a previous report using cell-based assays. Effective permeability coefficients (Peff) were determined in rats for 14 compounds, and ranged from 0.043x 10(-4) cm s(-1) to 1.67 x 10(-4) cm s(-1). These values strongly correlated (r2 = 0.88) with reported Peff values for man. In addition, the Spearman rank correlation coefficient calculated for in-situ-derived Peff and absorption in man was 0.92 while for the previously tested in-vitro Caco-2 and MDCK systems vs absorption in man, the correlation coefficients were 0.61 and 0.59, respectively. SPIP provided a better prediction of human absorption than the cell-based assays. This method, although time consuming, could be used as a secondary test for studying the mechanisms governing the absorption of new compounds, and for predicting more accurately the fraction absorbed in man.
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Affiliation(s)
- L Salphati
- Affymax Research Institute, Santa Clara, CA 95051, USA.
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Tamai I, Sai Y, Ono A, Kido Y, Yabuuchi H, Takanaga H, Satoh E, Ogihara T, Amano O, Izeki S, Tsuji A. Immunohistochemical and functional characterization of pH-dependent intestinal absorption of weak organic acids by the monocarboxylic acid transporter MCT1. J Pharm Pharmacol 1999; 51:1113-21. [PMID: 10579682 DOI: 10.1211/0022357991776804] [Citation(s) in RCA: 86] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022]
Abstract
The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues. Immunohistochemical analysis by use of the anti-MCT1 antibody showed that MCT1 is distributed throughout the upper and lower intestines, especially in the basolateral membrane and, to a lesser extent, in the brush-border membrane. When the transporter gene rat MCT1 was transfected into MDA-MB231 cells, transport of benzoic acid, a model weak organic acid that has been generally believed to be transported across the cell membranes by passive diffusion, and lactic acid in rat MCT1-transfected cells was significantly increased compared with transport in cells transfected with the expression vector pRc-CMV alone (mock cells). The observed transport was pH-dependent and activity increased between pH 7.5 and pH 5.5, whereas pH-dependence in mock cells was moderate. Rat MCT1-mediated benzoic acid uptake was saturable, with an apparent Km value of 3.05 mM. In addition, MCT1 increased the efflux of [14C]benzoic acid from the cells. Several weak organic acids were also transported by rat MCT1. These results show that pH-dependent intestinal absorption of weak organic acids, previously explained in terms of passive diffusion according to the pH-partition hypothesis, is at least partially accounted for by MCT1-mediated transport energized at acidic pH by utilization of the proton gradient as a driving force.
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Affiliation(s)
- I Tamai
- Faculty of Pharmaceutical Sciences, Kanazawa University, Japan
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Iseki K, Hirano T, Tsuji K, Miyazaki S, Takada M, Kobayashi M, Sugawara M, Miyazaki K. Ionic-diffusion potential-dependent transport of a new quinolone, sparfloxacin, across rat intestinal brush-border membrane. J Pharm Pharmacol 1998; 50:627-34. [PMID: 9680072 DOI: 10.1111/j.2042-7158.1998.tb06896.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The mechanism of uptake of sparfloxacin, a new quinolone, by intestinal brush-border membrane vesicles was investigated to clarify whether there is a common transport process for new quinolones mediated by the diffusion potential across the intestinal membrane bilayer. Sparfloxacin was taken up pH-dependently by rat intestinal brush-border membrane vesicles, behaviour analogous to that of organic cations including enoxacin and ciprofloxacin. Transient overshooting uptake of this quinolone was observed in the presence of an outward H+ gradient. Momentary dissipation of the H+ gradient by addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone did not affect the uptake of sparfloxacin, and a marked but incomplete reduction in the H+-sensitive overshooting uptake of sparfloxacin was apparent in the voltage-clamped brush-border membrane vesicles. Furthermore, a valinomycin-induced K+-diffusion potential (interior negative) and an inward C1--diffusion potential stimulated the initial uptake of sparfloxacin at pH 5.5. Sparfloxacin uptake was inhibited by tetracaine and imipramine. The inhibitory effect of these cations correlated well with changes in membrane surface charges induced by the presence of tetracaine or imipramine. These results indicate that sparfloxacin transport across the brush-border membrane depends upon the inside-negative ionic diffusion potential, that the H+- or K+-diffusion-potential-dependent uptake of sparfloxacin by intestinal brush-border membrane vesicles is affected by the membrane surface potential and that inhibition of sparfloxacin uptake originates from changes in the membrane surface potential caused by the organic cations.
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Affiliation(s)
- K Iseki
- Department of Pharmacy, Hokkaido University Hospital, School of Medicine, Hokkaido University, Sapporo, Japan
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Abstract
This review focuses on permeability measurements in humans, briefly discussing different perfusion techniques, the relevance of human Peff values, and various aspects of in vivo transport mechanisms. In addition, human Peff values are compared with corresponding data from three preclinical transport models. The regional human jejunal perfusion technique has been validated in several important ways. One of the most important findings is that there is a good correlation between the measured human effective permeability values and the extent of absorption of drugs in humans determined by pharmacokinetic studies. Estimations of the absorption half-lives from the measured Peff agree very well with the time to maximal amount of the dose absorbed achieved after an oral dose in humans. We have also shown that it is possible to determine the Peff for carrier-mediated transported compounds and to classify them according to the proposed biopharmaceutical classification system (BCS). Furthermore, human in vivo permeabilities can be predicted using preclinical permeability models, such as in situ perfusion of rat jejunum, the Caco-2 model, and excised intestinal segments in the Ussing chamber. The permeability of passively transported compounds can be predicted with a particularly high degree of accuracy. However, special care must be taken for drugs with a carrier-mediated transport mechanism, and a scaling factor has to be used. Finally, the data obtained in vivo in humans emphasize the need for more clinical studies investigating the effect of physiological in vivo factors and molecular mechanisms influencing the transport of drugs across the intestinal and as well as other membrane barriers. It will also be important to study the effect of antitransport mechanisms (multidrug resistance, MDR), such as efflux by P-glycoprotein(s) and gut wall metabolism, for example CYP 3A4, on bioavailability.
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Affiliation(s)
- H Lennernäs
- Department of Pharmacy, Group of Biopharmaceutics, Uppsala University, Sweden.
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Imanidis G, Waldner C, Mettler C, Leuenberger H. An improved diffusion cell design for determining drug transport parameters across cultured cell monolayers. J Pharm Sci 1996; 85:1196-203. [PMID: 8923325 DOI: 10.1021/js960102g] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
An improved two-chamber diffusion cell was developed for the study of drug transport across cultured cell monolayers. The cell monolayer was grown on a horizontal support membrane of polycarbonate, which could be rotated providing theoretically predictable thicknesses of the diffusion boundary layer in the donor and the receiver solution as a function of rotation rate. Permeation measurements were performed using the support membrane with and without a cell monolayer. The Madin-Darby-Bovine-Kidney (MDBK) cell line was employed, and permeability coefficients of model solutes (salicylic acid, mannitol, testosterone) across the three distinct mass transport barriers (i.e., the cell monolayer, the support membrane, and the diffusion boundary layer) were determined. The permeability of the diffusion boundary layer followed the theoretical dependence on the rotation rate; absolute values, however, deviated from predictions. Permeability coefficients for all three transport barriers varied substantially between solutes. This variation was the strongest for the permeability coefficient for the cell monolayer and resulted in a varying relative significance of these three barriers in controlling permeation kinetics. This improved diffusion apparatus permits the measurement of unbiased permeability values of solutes across the cell monolayer, notably when the cell monolayer is not absolutely the rate-determining barrier.
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Affiliation(s)
- G Imanidis
- School of Pharmacy, University of Basel, Switzerland
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Abstract
Recent advances in the field of carrier-mediated intestinal absorption of of amino acids, oligopeptides, monosaccharides, monocarboxylic acids, phosphate, bile acids and several water-soluble vitamins across brush-border and basolateral membranes are summarized. An understanding of the molecular and functional characteristics of the intestinal membrane transporters will be helpful in the utilization of these transporters for the enhanced oral delivery of poorly absorbed drugs. Some successful examples of the synthesis of prodrugs recognized by the targeted transporters are described. Functional expression of the multidrug resistance gene product, P-glycoprotein, as a primary active transporter in the intestinal brush-border membrane leads to net secretion of some drugs such as anticancer agents in the blood-to-luminal direction, serving as a secretory detoxifying mechanism and as a part of the absorption barrier in the intestine.
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Affiliation(s)
- A Tsuji
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan
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38
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39
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Fagerholm U, Lennernäs H. Experimental estimation of the effective unstirred water layer thickness in the human jejunum, and its importance in oral drug absorption. Eur J Pharm Sci 1995. [DOI: 10.1016/0928-0987(95)00027-b] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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Sugawara M, Hashimoto A, Kobayashi M, Iseki K, Miyazaki K. Effect of membrane surface potential on the uptake of anionic compounds by liposomes. BIOCHIMICA ET BIOPHYSICA ACTA 1994; 1192:241-6. [PMID: 7517186 DOI: 10.1016/0005-2736(94)90124-4] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The effect of membrane surface potential on the uptake of several anionic compounds by liposomes (large unilamellar vesicles), which contain various amounts of dipalmitoylphosphatidylserine (DPPS), was investigated. The uptake amount of four tested anionic compounds (cefixime, benzyloxyindoleacetic acid (BOIAA), ceftibuten and S-1006) decreased with an increase in the DPPS content of liposomes, and was correlated with the membrane surface potential monitored using a fluorescent dye, 8-anilino-1-naphthalene sulfonate (ANS). Moreover, for all of the tested anionic compounds, a good correlation was observed between the ratio of the uptake value (5 min) by each of the liposomes comprising various amounts of DPPS to the uptake value by liposomes containing 10% DPPS and a relative membrane surface potential monitored by ANS. On the other hand, the uptake of zwitterionic compounds (enoxacin, cephradine and benzyloxytryptophan (BOTP)) was independent of DPPS content. These results suggest that the uptake of tested anionic compounds by large unilamellar lipid vesicles is dependent on the membrane surface potential which originates in the surface negative charge.
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Affiliation(s)
- M Sugawara
- Department of Pharmacy, Hokkaido University Hospital, School of Medicine, Hokkaido University, Sapporo, Japan
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41
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Sugawara M, Hashimoto A, Toda T, Takahashi M, Kobayashi M, Iseki K, Miyazaki K. Changes in the permeation rate of organic anions through the intestinal brush-border membrane with membrane surface potential. BIOCHIMICA ET BIOPHYSICA ACTA 1994; 1190:85-90. [PMID: 8110822 DOI: 10.1016/0005-2736(94)90036-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The effects of membrane surface potential on the uptake of anionic compounds by rat intestinal brush-border membrane vesicles were investigated. The uptake amount of all tested anionic compounds (ceftibuten, cefixime, benzylpenicillin, s-1006 and rentiapril) in the neutral medium (pH 7.5) was lower than that in the acidic medium (pH 5.5). Changes in surface potential of brush-border membrane vesicles were monitored using a fluorescence dye, 8-anilino-1-naphthalenesulfonate (ANS), and the results suggested an increase of a negative charge on the membrane surface proportional to the increase of the pH of medium. A good correlation was observed between the initial uptake rate of all tested anionic compounds and relative membrane surface potential monitored by ANS. Moreover, the uptake of cefixime by artificial liposome made from PC containing various amount of DPPS was measured. The uptake value of cefixime was decreased in proportion to an increase of DPPS content. These results suggest that the permeation of anionic compounds across intestinal brush-border membrane is dependent on surface potential originate in the surface negative charge.
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Affiliation(s)
- M Sugawara
- Department of Pharmacy, Hokkaido University Hospital, School of Medicine, Hokkaido University, Sapporo, Japan
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42
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Tsuji A, Takanaga H, Tamai I, Terasaki T. Transcellular transport of benzoic acid across Caco-2 cells by a pH-dependent and carrier-mediated transport mechanism. Pharm Res 1994; 11:30-7. [PMID: 8140053 DOI: 10.1023/a:1018933324914] [Citation(s) in RCA: 117] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
The pH-dependent transcellular transport of [14C]benzoic acid across a Caco-2 cell monolayer is shown to be mediated by a monocarboxylic acid-specific carrier-mediated transport system, localized on the apical membrane. Evidence for the carrier-mediated transport of benzoic acid includes (a) the significant temperature and concentration dependence, (b) the metabolic energy dependence, (c) the inhibition by unlabeled benzoic acid and other monocarboxylic acids, (d) countertransport effects on the uptake of [14C]benzoic acid, and (e) effects of a proteinase (papain) and amino acid-modifying reagents. Furthermore, since carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin significantly inhibited the transport of [14C]benzoic acid, the direct driving force for benzoic acid transport is suggested to be the inwardly directed proton gradient. From these results, together with previous observations using intestinal brush border membrane vesicles, the pH dependence of the transcellular transport of certain organic weak acids across Caco-2 cells is considered to result mainly from a proton gradient-dependent, carrier-mediated transport mechanism, rather than passive diffusion according to the pH-partition theory.
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Affiliation(s)
- A Tsuji
- Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan
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43
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Abstract
The rate of F absorption from the stomach is pH-dependent, with greater absorption at low pH. Since the rate of absorption is also strongly influenced by the rapidity of gastric emptying, we have compared the relative importance of gastric acidity and gastric emptying in overall F absorption. Male rats (350 g, n = 85) were pre-treated with cimetidine (to inhibit gastric acid secretion) or pentagastrin (to stimulate gastric acid secretion) or were untreated controls, and given 50 micrograms F by stomach intubation. The pH of the F-containing solution was varied in the cimetidine-pre-treated group (pH 1.5, 5.5, 8.5), and was 5.5 for the control and pentagastrin-pre-treated groups. Gastric emptying was measured by addition of 14C polyethylene glycol to the F solution as an unabsorbed marker of fluid movement. F absorption was measured after 10, 20, and 40 min. The rate of gastric emptying was unaffected by pre-treatment or pH of the intubating solution. Initially, F absorption was greatest at low pH. After 40 min, absorption was comparable in all groups, averaging approximately 70% of the initial dose. The extent of absorption from the stomach was inversely related to pH, but increased absorption from the small intestine compensated for the low gastric absorption at high pH.
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Affiliation(s)
- H H Messer
- School of Dentistry, University of Minnesota, Minneapolis 55455
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44
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Roig T, Vinardell MP. Intestinal perfusion in vivo for the study of absorptive processes. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY. A, COMPARATIVE PHYSIOLOGY 1991; 98:3-7. [PMID: 1673373 DOI: 10.1016/0300-9629(91)90568-w] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
1. Intestinal absorption can be studied by in vitro and in vivo techniques. Among the in vivo ones, intestinal perfusion is the one more employed. 2. Intestinal perfusion could be performed by a simple perfusion of an intestinal segment or by a double perfusion of the intestine and the vascular bed simultaneously. 3. The double perfusion has the advantage of measuring the substrate appearance in the vascular circuit. 4. In this review we compare the different techniques described in the literature, paying attention to their advantages. 5. The best method is the one that maintains the animal alive throughout the experiment, because it provides information about intestinal absorption under conditions similar to the natural ones.
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Affiliation(s)
- T Roig
- Department de Ciències Fisiologiques Humanes i de la Nutrició, Facultat de Farmàcia, Universitat de Barcelona, Spain
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45
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McEwan GT, Lucas ML. The effect of E. coli STa enterotoxin on the absorption of weakly dissociable drugs from rat proximal jejunum in vivo. Br J Pharmacol 1990; 101:937-43. [PMID: 2085716 PMCID: PMC1917823 DOI: 10.1111/j.1476-5381.1990.tb14184.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
1. The effect of E. coli heat stable (STa) enterotoxin on the absorption of radio-labelled weak electrolytes and their appearance in peripheral blood was assessed in vivo by use of an intestinal recirculation procedure. 2. STa reduced the luminal disappearance (P less than 0.02) and peripheral blood appearance (P less than 0.02) of label from salicylic acid as well as the luminal disappearance (P less than 0.02) of diphenylhydantoin. 3. In contrast, STa increased the appearance in peripheral blood and disappearance from the lumen of label from morphine (P less than 0.05), amphetamine (P less than 0.01) and lignocaine (P less than 0.01). 4. Increased weak base (lignocaine) absorption can also be achieved by a combination of forskolin and theophylline which resembles STa in its ability to neutralise the usually acid surface pH of the proximal jejunum. 5. Increased weak base absorption and hindered weak acid absorption occurs despite a uniform reduction in net fluid absorption after STs exposure, making it unlikely that variations in fluid absorption account for the variations in drug absorption. 6. The ability of STa to elevate the mucosal surface pH (or acid microclimate) to neutral values, thereby altering the proportion of uncharged weak-electrolyte, may explain its different effects on weak acids and bases: neutralisation of the acid microclimate would increase the amount of undissociate weak base available for uptake.
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Affiliation(s)
- G T McEwan
- Institute of Physiology, University of Glasgow, Scotland
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46
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47
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McEwan GT, Schousboe B, Skadhauge E. Direct measurement of mucosal surface pH of pig jejunum in vivo. ZENTRALBLATT FUR VETERINARMEDIZIN. REIHE A 1990; 37:439-44. [PMID: 2120867 DOI: 10.1111/j.1439-0442.1990.tb00926.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The mucosal surface pH of pig jejunum was measured in vivo. When the pH in the bulk phase of the perfusing buffer solution was 7.10, the pH at the mucosa (as measured by a miniaturised glass pH electrode) was 6.19 +/- 0.04 (n = 19). This relatively acidic mucosal surface pH is not an anoxic artefact since anoxia resulted in a significant alkalinisation of the mucosa. This finding, that the pig, like man and the rat, has a jejunal "acid microclimate" has important implications for weak electrolyte absorption from the upper small intestine of this species as well as for any other pH dependent brush border process.
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Affiliation(s)
- G T McEwan
- Dept. of Physiological Sciences, Medical School, Univ. of Newcastle upon Tyne, England, U.K
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48
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Böttger WM, Schoonen AJ, de Vries-Nijboer GW, Visser J, Meijer DF. The influence of pH on rectal absorption of sodium benzoate studied in man by rectal lumen perfusion. JOURNAL OF PHARMACOKINETICS AND BIOPHARMACEUTICS 1990; 18:1-15. [PMID: 2329467 DOI: 10.1007/bf01063619] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The influence of pH on rectal absorption of sodium benzoate in man was studied by means of a rectal lumen perfusion method and compared with in vitro measurements on diffusional transport of sodium benzoate across an octanol/water interface. For nonbuffered solutions of benzoate in vitro, it was shown that mass flux across an octanol/water interface occurs in agreement with the pH-partition model. In vivo however, mass flux increases less with decreasing pH of unbuffered perfusate than is anticipated on the basis of the pH-partition model. Probably an alkaline flow across the rectal mucosa into the lumen is present as a physiological neutralization mechanism. In contrast, buffered solutions of benzoate show a linear relationship between mass flux and decreasing pH in vitro as well as in vivo. The effect of buffer on the concentration profile of benzoic acid is qualitatively explained. It is shown that an alkaline flow across the rectal mucosa only slightly influences absorption of benzoic acid from strongly buffered solutions in the rectal lumen. It is concluded that the use of strong buffers in rectal solutions induces a drastic effect on the pH of the boundary layer, an effect not seen for unbuffered solutions. This phenomenon does not invalidate the pH-partition hypothesis but can be explained by it.
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Affiliation(s)
- W M Böttger
- St. Maartens Gasthuis Hospital, Pharmaceutical Department, Venlo, The Netherlands
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49
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Iwatsubo T, Yamazaki M, Sugiyama Y, Suzuki H, Yanai S, Kim DC, Satoh H, Miyamoto Y, Iga T, Hanano M. Epidermal growth factor as a regulatory hormone maintaining a low pH microclimate in the rat small intestine. J Pharm Sci 1989; 78:457-9. [PMID: 2788214 DOI: 10.1002/jps.2600780606] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
This study was designed to determine the effect of epidermal growth factor (EGF) in the lumen on the pH of the intestinal surface in the rat jejunum, which is referred to as "microclimate-pH". In the control experiment, a significant pH gradient was observed between the mucosal surface (approximately pH 6.8) and the bulk phase (approximately pH 7.3). The microclimate-pH was decreased by 0.2-0.6 pH units after addition of higher concentrations of EGF (3-100 nM) to the lumen. The microclimate-pH thus decreased recovers to the control value by replacing EGF with TES buffer, suggesting that the EGF effect is reversible. Considering that the Na+-H+ exchanger exists on the luminal membrane of the intestinal cells, the decrease in the microclimate-pH which was induced by EGF added to the luminal side may be due to the activation of Na+-H+ exchanger.
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Affiliation(s)
- T Iwatsubo
- Faculty of Pharmaceutical Sciences, University of Tokyo, Japan
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50
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McKie AT, Kusel M, McEwan GT, Lucas ML. The effect of heat-stable Escherichia coli enterotoxin, theophylline and forskolin on cyclic nucleotide levels and mucosal surface (acid microclimate) pH in rat proximal jejunum in vivo. BIOCHIMICA ET BIOPHYSICA ACTA 1988; 971:325-31. [PMID: 2844294 DOI: 10.1016/0167-4889(88)90148-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The normally acidic mucosal surface pH of 6.24 +/- 0.02(30) in rat proximal jejunum in vivo is effectively neutralised by 30 min exposure to heat-stable Escherichia coli (STa) enterotoxin (14 micrograms/ml) to 6.80 +/- 0.07 (n = 5) or to a forskolin/theophylline combination (1 mM:20 mM) to 7.10 +/- 0.07(7) while perfusion with Krebs-phosphate buffer alone without glucose left the mucosal surface pH unchanged at a pH of 6.21 +/- 0.02(9). Forskolin alone had no effect, and 20 mM theophylline moderately elevated the surface pH to 6.52 +/- 0.03(5). Theophylline, forskolin and their combination all elevated cAMP levels per mg tissue DNA above control values while STa enterotoxin was without effect. In contrast, all agents elevated cGMP levels per mg tissue DNA above control levels. These findings indicate that surface pH is only moderately affected by changes in cAMP levels and is affected to a much greater extent by altered cGMP levels.
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Affiliation(s)
- A T McKie
- Institute of Physiology, Glasgow University, U.K
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